Inhibition of the SGLT2/NHE-1/NLRP3 signaling axis attenuates neuroinflammation and oxidative stress to ameliorate seizures and cognitive impairment in epileptic mice.

OBJECTIVE

Beyond neuroinflammation, oxidative stress is a key pathomechanism in epilepsy. This study investigated a novel SGLT2/NHE-1/NLRP3 signaling axis and evaluated its role in driving oxidative stress and neuroinflammation in epilepsy. We aimed to determine whether targeted inhibition of this axis could alleviate neuronal excitability and cognitive deficits by restoring redox balance and suppressing neuroinflammation.

METHODS

Network pharmacology predicted the primary anti-epileptic target of dapagliflozin. Bioinformatic analysis was performed on the GEO dataset GSE256068 from patients with temporal lobe epilepsy. A PTZ-kindled mouse model was established and treated with dapagliflozin (SGLT2 inhibitor), cariporide (NHE-1 inhibitor), or CY09 (NLRP3 inhibitor). Seizure behavior and EEG were recorded; cognitive function was assessed using the Morris water maze. Molecular analyses (RT-qPCR, Western blot, immunohistochemistry, ELISA, etc.) were conducted to evaluate neuroinflammation and oxidative stress. Complementary in vitro studies used HT22 hippocampal neuronal cells (a glia-free model) to validate the neuron-intrinsic operation of axis's role; Targeted inhibition of each component was performed using specific inhibitors, and molecular interactions were interrogated through overexpression, functional rescue experiments, molecular docking, and co-immunoprecipitation.

RESULTS

Bioinformatic and molecular analyses confirmed concerted upregulation of SGLT2, NHE-1, and NLRP3 in epileptic human and mouse hippocampi (p < 0.01), with significant enrichment in NOD-like receptor signaling pathway. All three inhibitors not only reduced seizure severity included seizure scores (mean seizure grade decreased from 4.88 to 3.12-3.48, p < 0.01), abnormal EEG discharges, and seizure duration (mean duration decreased from 42.32 min to 5.33-9.77 min, p < 0.01), but aslo improved spatial learning and memory abilities. In addition, inhibition of SGLT2/NHE-1/NLRP3 signaling axis mitigated oxidative damage by reducing ROS production and lipid peroxidation, while enhancing antioxidant defense (p < 0.05). Crucially, they suppressed NLRP3 inflammasome activation and neuroinflammation. In vitro studies defined a core unidirectional SGLT2→NHE-1→NLRP3 cascade functioning within neurons and revealed its operation within a self-amplifying regulatory network, demonstrating that inhibition at any node effectively attenuated both LPS-induced oxidative stress and inflammatory responses in the absence of glial cells. Direct protein interactions within the axis were identified, supporting the formation of a functional signaling complex. This integrated model positions oxidative stress as both a trigger and a sustained component coupled with neuroinflammation in a feed-forward loop.

CONCLUSION

Our findings unveil the SGLT2/NHE-1/NLRP3 axis as a master regulator that integrates oxidative stress and neuroinflammation in epilepsy. Notably, we identify this as a neuron-intrinsic pathway, and its targeted inhibition of this axis restores redox homeostasis and attenuates neuroinflammation, leading to improved seizure control and cognitive function. This study provides a novel mechanistic rationale for repurposing SGLT2 inhibitors as a multi-target therapy against epilepsy-related oxidative damage and neuroinflammation.